An exploratory human study of superstable homogeneous lipiodol–indocyanine green formulation for precise surgical navigation in liver cancer

Abstract The clinical applications of transcatheter arterial embolization (TAE) conversion therapy combined with hepatectomy have been severely restricted by ill‐defined tumoral boundaries and miniscule hidden lesions. Fluorescent surgical navigation is a promising method for overcoming these barriers. However, sufficient delivery of the fluorescent probe into the tumor region after long‐term TAE is challenging due to blockade of the tumor‐supplying artery. Here, a super‐stable homogeneous intermix formulating technology (SHIFT) to physically mix lipiodol and indocyanine green (ICG) formulation (SHIFT and ICG) for fluorescent surgical navigation after long‐term TAE conversion therapy is provided. Through the retrospective study of 45 clinical liver cancer patients, it is found that SHIFT and ICG formulation have excellent tumor deposition effect and safety. During surgical resection after long‐term TAE conversion therapy, SHIFT and ICG could clearly identify in real time the full tumor regions and boundaries and had a high signal‐to‐normal tissues ratio—even the indistinguishable satellite lesions could be identified with a strong fluorescence intensity. Meanwhile, SHIFT and ICG could improve operative, anesthetic, and postoperative variables associated with postoperative complications. This simple and effective SHIFT could provide precise fluorescent navigation for surgical resection following long‐term embolization therapy in clinical practice and has great potential for a translational pipeline.


Abstract
The clinical applications of transcatheter arterial embolization (TAE) conversion therapy combined with hepatectomy have been severely restricted by ill-defined tumoral boundaries and miniscule hidden lesions. Fluorescent surgical navigation is a promising method for overcoming these barriers. However, sufficient delivery of the fluorescent probe into the tumor region after long-term TAE is challenging due to blockade of the tumor-supplying artery. Here, a super-stable homogeneous intermix formulating technology (SHIFT) to physically mix lipiodol and indocyanine green (ICG) formulation (SHIFT and ICG) for fluorescent surgical navigation after long-term TAE conversion therapy is provided. Through the retrospective study of 45 clinical liver cancer patients, it is found that SHIFT and ICG formulation have excellent tumor deposition effect and safety. During surgical resection after long-term TAE conversion therapy, SHIFT and ICG could clearly identify in real time the full tumor regions and boundaries and had a high signalto-normal tissues ratio-even the indistinguishable satellite lesions could be identified with a strong fluorescence intensity. Meanwhile, SHIFT and ICG could improve operative, anesthetic, and postoperative variables associated with post-Pan He, Yongfu Xiong, Bin Luo, and Jianming Liu contributed equally to this work.

| INTRODUCTION
Liver cancer, the sixth most prevalent malignancy worldwide, is characterized by poor prognosis and high mortality rates. 1 Radical resection remains the first line of treatment for liver cancer. 2 However, due to the disease's rapid progression, many patients lost the opportunity for initial surgical resection when seeking treatment. Direct resection in cases in which there is a large tumor volume or a heavy tumor burden can cause excessive trauma, while the insufficient residual liver volume may lead to postoperative liver failure. [3][4][5] Therefore, to prolong relapse-free and overall survival, it is essential to improve the overall prognosis of liver cancer by transforming initially unresectable patients into operable patients, palliative surgery into a radical resection, and the R1 resection into an R0 resection. 6,7 The concept of conversion therapy was first proposed in 1977 by Shafer and Selinkoff. 8 Transcatheter arterial embolization (TAE) is the most common method of conversion therapy; it embolizes the tumor's blood supply arteries to control tumor growth and reduces the proliferation of cancer cells caused by intraoperative extrusion for the improvement of radical surgical resection rate. [9][10][11] However, during surgical resection, identifying small lesions and tumor boundaries is often difficult, leading to residual liver cancer tissues and postoperative tumor recurrence, 12,13 insufficient residual liver volume, and a hindered recovery of liver function after hepatectomy. 14 Therefore, molecular imaging must be developed to distinguish boundaries and small tumor lesions for accurate complete resection of tumor lesions with minimal trauma.
Indocyanine green (ICG) is a near-infrared fluorescent dye. Given its excellent optical properties and biosafety in vivo, ICG has been used in fluorescence-guided surgical resection and intraoperative tumor visualization. [15][16][17] However, in the interventional embolization of conversion therapy for liver cancer, in cases in which ICG-a small molecule-is injected intravenously (i.v.) prior to tumor embolization, the limited stability, ease of metabolism, and agglomeration quenching issues of ICG lead to poor fluorescence performance. 18 Administration of i.v. ICG after tumor embolization prevents the infiltration and distribution of ICG in the tumor tissues that results from the blocked supply artery and tumor necrosis. 19,20 Therefore, surgical resection after interventional embolization of conversion therapy cannot be effectively and precisely navigated in the clinic.
To address this issue, we developed a super-stable homogeneous intermix formulating technology (SHIFT) to physically mix lipiodol and ICG formulations (SHIFT and ICG) homogeneously for fluorescencenavigated hepatectomy after long-term TAE conversion therapy, which realized the interventional embolization with one-time administration and follow-up surgical fluorescence navigation. Previous work has confirmed that SHIFT formulation was prepared in a green physical mixture via a carrier-free manner, which possesses controlled morphology, long-term stability, and improved optical characteristics of ICG. Furthermore, the viscosity of the SHIFT and ICG was comparable to lipiodol and exhibited long-lasting excellent fluorescence navigation in VX2 orthotopic hepatocarcinoma models. 21 To elaborate on the results of the superior performance of SHIFT and ICG previously reported, 22,23 we conducted a clinical application trial. The SHIFT and ICG formulation prepared via SHIFT performs excellent tumor-specific deposition after long-term TAE therapy, which enables ICG to continuously illuminate the tumor regions and microsatellite lesions for specific resection (Figure 1). SHIFT and ICG assist surgeons at distinguishing proper lesions and determining the accurate dissecting plane during the entire hepatectomy, which, therefore, could provide a sound option for high-precision, real-time navigation of liver cancer surgery. The clinical data of 20 liver cancer patients who received TAE with conventional lipiodol as the main embolic agent were used as a control group. We conducted a retrospective analysis of the clinicopathological data to compare outcomes between the two groups. Exclusion criteria: (1) patients with recurrent disease or who were previously treated by surgery or drugs; (2) patients with other confirmed malignant tumors or serious diseases of the heart, lung, kidney, brain, and other important organs; and (3) those with an indocyanine green allergy or iodine allergy.

| Materials
Lipiodol was purchased from Jiangsu Hengrui Pharmaceutical Co, Ltd, Jiangsu, China. Indocyanine green was purchased from Dandong Medical and Pharmaceutical Co, Ltd, Dandong, China. An anti-Ki-67 antibody, F I G U R E 1 Schematic illustration of super-stable homogeneous intermix formulating technology (SHIFT) and indocyanine green (ICG) preparation and research process. First, (a) superstable homogeneous intermix formulation system was employed to produce SHIFT and ICG with enhanced imaging properties, anti-photobleaching capacity, and retention ability in tumor regions. Next, (b) liver cancer patients unable to be radically resected were enrolled in the study and received transcatheter arterial embolization (TAE) treatment with SHIFT and ICG as the main embolic agent. Subsequently, (c) the patients who reached the standard of radical surgical resection after TAE conversion therapy received a precise hepatectomy under real-time fluorescence. (d) After the operation, pathological examination of the resected tissues was conducted.

| Transarterial embolization and ICG administration
We punctured the femoral artery and performed celiac trunk and superior mesenteric artery angiography. After the tumor-supplying artery was identified, a microcatheter was used to super-selectively enter the target artery. First, local perfusion of the tumor was performed with lobaplatin for injection, and next an appropriate amount of SHIFT and ICG formulation (ICG concentration 1 mg/ml) (SHIFT and ICG group) or conventional lipiodol (control group) were injected respectively. The dosage of SHIFT and ICG formulation was injected according to the size of the tumor, generally within 10-20 ml, 25 until target artery blood stasis or regurgitation occurs and the embolization stops. 26 Finally, gelatine sponge particles (350-560 μm) sandwich method was used to strengthen embolization. Every 4 weeks followup reexamination after TAE was conducted to clarify the treatment

| Statistical analysis
Data analyzed by Prism 7 (GraphPad Software, San Diego, California) and SPSS 17.0 were presented as mean ± SD. The Mann-Whitney U test was used to evaluate the differences between the experimental group and the control group. p values < 0.05 were considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

| General information and baseline characteristics
In this study, 45 patients with unresectable liver cancer, including 29 men and 16 women, with a mean (±standard deviation) age of 57.05 ± 10.27 and 54.8 ± 12.14 years in the control and SHIFT and ICG group, respectively, were successfully treated with TAE, using conventional lipiodol or SHIFT and ICG formulation as the main embolization agents. An irregular liver surface due to cirrhosis was found in 16 patients. The mean ICG-R15 was 7.17% ± 2.58% and 7.28% ± 2.24% in the control and SHIFT and ICG group, respectively.
No intraoperative or postoperative adverse events were observed related to the administration of ICG. The mean tumor size was 9.63 ± 2.35 cm and 9.18 ± 2.06 cm in the control and SHIFT and ICG group, respectively. The basic characteristics and demographic information of both experimental groups are shown in Table 1, with no significant between-group differences identified. No obvious abnormality was found in the peripheral blood cells and blood coagulation indices of the two groups (Figure 3c,e,f).

|
Although alanine transaminase (ALT) and aspartate transaminase (AST) increased slightly on the third day after TAE in both groups, they returned nearly to normal on the seventh day (Figure 3d,g). This indicated that the SHIFT is only a physical process and does not alter the embolization performance of SHIFT and ICG. As a major embolization agent, SHIFT and ICG has an excellent safety profile.  (Figure 4l,m). These results suggest that after successful conversion therapy, the traditional i.v. ICG method is unable to provide effective and accurate navigation of surgical excision for liver cancer.

| The surgical navigation effect of SHIFT and ICG
Conversion therapy was successful in four patients treated with SHIFT and ICG for TAE, reaching the standard of radical surgical resection, as confirmed by CT ( Figure S1). Surgical resection under real-time fluorescence navigation was performed on these four patients. The CT imaging confirmed that SHIFT and ICG was fully deposited in the tumor area ( Figure S1). Subsequently, we further verified that the SHIFT and ICG could exert fluorescent navigation function after embolization (Figure 5a,b). The results indicated that SHIFT and ICG exhibited a superior fluorescent capability compared to controls, and before the tumor was resected, bright fluorescent signals were evenly distributed throughout the tumor lesions. The entire tumor lesions were illuminated (Figure 5c  To  (Table S1).
These results suggest that SHIFT and ICG is capable of rapid and accurate tumor identification and of reducing the risk of complications after hepatectomy. Although R0 resection was achieved in both groups, the surgical margin in the SHIFT and ICG group (19.75 ± 8.06 mm) was significantly smaller than that in the control group (40.67 ± 16.92 mm; p = 0.157; Figure 6f). Furthermore, analysis of the postoperative morbidity and mortality variables in both groups showed that one patient in the control group developed ascites, while no obvious abnormalities were found in the SHIFT and ICG group. Additionally, no deaths were reported in either group at 30 and 60 days post-surgery (Table S2).

| DISCUSSION
Liver cancer, which constitutes the most common tumor of the digestive system, is characterized by rapid onset, a high degree of malignancy, and rapid invasive growth; it is usually diagnosed in its middle and advanced stages. 28,29 Surgical resection is the first line of treatment 30 ; however, many patients are diagnosed too late for surgery, or when the tumor volume is too large. In these cases, direct resection can cause great trauma or residual tumor tissues with poor prognosis. These patients can be treated with secondary surgical resection after TAE or transcatheter arterial chemo-embolization of conversion therapy. 31,32 Unfortunately, identifying the boundary that delineates the tumor from normal liver tissue and microscopic lesions is difficult during surgical resection. Cancerous tissue often cannot be removed completely, leading to postoperative tumor recurrence. 33,34 Therefore, the development of an effective fluorescent molecular probe is urgently needed to distinguish the boundary between tumors and both normal tissue and microscopic lesions. 12,35 In clinical practice, lipiodol is a common agent in interventional embolization for liver cancer, due to its high viscosity and precise tumoral deposition. 36 However, sufficient delivery of the fluorescent probe into the tumor region after interventional embolization can prove challenging. Thus, we here present a strategy using a prepared fluorescent probe and lipiodol formulation for precise fluorescent surgical navigation after long-term TAE conversion therapy.
The ICG molecule is quickly metabolized, has photobleaching resistance and no targeting ability, and is insoluble in oily substances. 37 If fluorescence navigation is performed by traditional i.v.
injection after TAE treatment, it is highly difficult for ICG delivery in cancer tissues because the feeding artery of liver cancer is embolized.  Nevertheless, our work has some limitations, such as a small sample size, early staged patients, a rich blood supply to the tumor, and a lack of long-term outcomes data. These issues can be addressed by expanding the sample size, altering the inclusion criteria, and including close long-term follow-up; our future research aims to solve these issues. Furthermore, the SHIFT and ICG formulation can be extended to accurate, preoperative TAE-assisted fluorescence navigation for radical resection of large or small liver cancer tumors. The SHIFT drugs can be extended to chemotherapeutics and radiopharmaceuticals to develop enhanced chemotherapy and radiotherapy. 41,42

| CONCLUSIONS
In summary, such a green SHIFT and ICG formulation via SHIFT integrates the excellent stability, antiphotobleaching ability, imaging sensitivity, and specific tumoral deposition of lipiodol to address the clinical issues of fluorescent surgical navigation after long-term embolization therapy of conversion therapy.